Noninvasive monitoring of apoptosis versus necrosis in a neuroblastoma cell line expressing a nuclear pore protein tagged with the green fluorescent protein.
A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 microM staurosporine or 100 microM p-benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.
Imreh
G
G
Department of Biochemistry, Stockholm University, Stockholm, 106 91, Sweden.
Beckman
M
M
Iverfeldt
K
K
Hallberg
E
E
D017209:Apoptosis; D016227:Benzoquinones / Q000494:pharmacology; D015415:Biomarkers; D002843:Chromatin; D053938:DNA Fragmentation; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D009336:Necrosis; D009447:Neuroblastoma / Q000473:pathology*; D009685:Nuclear Envelope / Q000737:chemistry; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins / Q000032:analysis; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D016227:Benzoquinones; D015415:Biomarkers; D002843:Chromatin; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C082264:Pom121 protein, rat; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C004532:quinone; D019311:Staurosporine